Versatility of protein-protein interactions with Mediator in different biological processes

Abstract

The Mediator complex plays a crucial role in transcription as a co-activator that regulates the expression of most RNA Polymerase II (Pol II) transcripts. Beyond its role in recruiting Pol II and the general transcription factors to form the Pre-initiation complex (PIC), Mediator has also been shown to impact the state of the chromatin and plays a role in the genomic organization. It also mediates functions such as DNA repair or RNA-splicing. It is still unknown how this protein complex manages multiple operations efficiently and can interact with an extremely high number of proteins inside the nucleus. How the Mediator complex establishes low affinity and high specificity protein-protein interactions is the focus of this study. The role of Mediator in transcription is vast; here, we focus on a specific interaction between Mediator and the transcription factor SOX18, whose role is crucial for cell fate in early embryogenesis. We unravelled how this TF interacts with the Tail module of Mediator and how this interaction is restricted to the transactivation domain of the protein. We used single-molecule techniques to reveal these protein-protein interactions via AlphaScreen proximity assay between SOX18 WT and synthetic constructs containing different regions of the protein: we also studied the interactions between Mediator and SOX18 pathological mutants. These mutations lead to the development of a disease called HLTRS, in which patients present anomalies in the development of their blood vessels and lymphatic system. It was crucial to understand how those mutations can affect the interactions with Mediator. We discover that mutations associated with truncations losing the TAD domain have an entirely different interaction pattern than those still conserving a portion. This study also touches on the role of Mediator and DNA repair via two specific subunits Med17 and Med20. Specifically, Med20 was described as presenting a series of mutants that leads to a disease presenting phenotypes comparable to Cockayne’s syndrome, a disease caused by DNA repair deficiencies. We study the PPIs within those mutants, and DNA repair proteins from four different DNA repair pathways (NER, NER-GG, NER-TCR and NEHJ); our results revealed that these mutations do not seem to affect the interactions with DNA repair proteins dramatically, but this appears to be sufficient to impair DNA repair. Thermostability assays against Med20 mutants and Med20 WT revealed similar behaviour within missense mutants, but early protein aggregation on the mutation led to a truncation. Finally, we investigated the role of virulent agents against Mediator: since Mediator is a multifaceted complex in charge of several essential functions on the cell, could viruses target them? We tested the ability of the two main proteases of SARS-CoV-2, Nsp3 and Nsp5, to cleave Mediator subunits in vitro. We discovered that out of 26 subunits tested, Nsp5 could cleave four (Med15, Med21, Med23 and Med28), and Nsp3 could cleave Med16, although no evidence was related Nsp3 to the nucleus as Nsp5. One of the cleaved proteins, Med23, acts as a coactivator of the signalling pathway WNT/β catenin and could significantly affect disease progression.